Project 1 consists of three subprojects. Subproject 1 will test the hypothesis that iron is transported across erythroid mitochondrial membranes as Fe(II). A ferroxidase is required on the inner mitochondrial membrane for effective iron uptake. Ferrochelatase is likely the required ferroxidase. This hypothesis will be tested with isolated mitochondria obtained from induced MEL cells. Genes responsible for erythroid-specific mitochondrial iron transport will be cloned and characterized and the effects of mutant ferrochelatase will be determined. Subproject 2 will define the properties of two iron regulatory element binding proteins, IRE-BP1 and IRE-BP2. The role of each of these proteins will be determined by targeted gene disruptions in ES cells and in transgenic animals. Subproject 3 will result in the creation of a model of human familial porphyria cutanea tarda (PCT) by targeted disruption of the uroporphyrinogen decarboxylase (URO-D) gene and the creation of transgenic animals heterozygous for the disruption. These animals will be used to test the hypothesis that phenotypic expression of PCT is dependent on a P450-mediated reaction which oxidizes the substrate of URO-D.